Immune Netw.  2009 Dec;9(6):274-284. 10.4110/in.2009.9.6.274.

Swiprosin-1 Regulates Cytokine Expression of Human Mast Cell Line HMC-1 through Actin Remodeling

• Ramesh TP
• Kim YD
• Kwon MS
• Jun CD
• Kim SW

• KMID: 2150656
• DOI: http://doi.org/10.4110/in.2009.9.6.274

Abstract

BACKGROUND
Swiprosin-1 was identified in human CD8+ lymphocytes, mature B cells and non-lymphonoid tissue. We have recently reported that swiprosin-1 is expressed in mast cells and up-regulated in both in vitro and in vivo. METHODS: The expression of cytokines and swiprosin-1 were determined by by real time PCR and conventional PCR. Pharmacological inhibitors were treated to investigate potential mechanism of swiprosin-1 in mast cell activation. Actin content was evaluated by confocal microscopy and flow cytometry. RESULTS: The swiprosin-1 augmented PMA/A23187-induced expression of cytokines and release of histamine. However, knock-down of swiprosin-1 showed only a modest effect on PMA/A23187-induced cytokine expression, suggesting that swiprosin-1 has gain-of-function characteristics. Swiprosin-1 was found in microvilli-like membrane protrusions and highly co-localized with F-actin. Importantly, either disruption of actin by cytochalasin B or inhibition of PI3 kinase, an enzyme involved in actin remodeling, by wortmannin blocked cytokine expression only in swiprosin-1-overexpressing cells. CONCLUSION: These results suggest that swiprosin-1 modulates mast cell activation potentially through actin regulation.

Keyword

Mast cells; Swiprosin-1; Protein kinase C; Actin remodeling

MeSH Terms

Actins
Androstadienes
B-Lymphocytes
Cytochalasin B
Cytokines
Flow Cytometry
Histamine
Humans
Lymphocytes
Mast Cells
Membranes
Microscopy, Confocal
Phosphotransferases
Polymerase Chain Reaction
Protein Kinase C
Real-Time Polymerase Chain Reaction
Actins
Androstadienes
Cytochalasin B
Cytokines
Histamine
Phosphotransferases
Protein Kinase C

Figure

• 

  Figure 1 Expression analysis of swiprosin-1 mRNA by RT-PCR. (A) Expression of swiprosin-1 mRNA in various cell types. RNAs were prepared from cell lines including Jurkat T and Molt-4 T (T cells), Raji B (B cells), THP-1 (monocytes), 293T, CHO-K1, HT-29 (epithelial cells), and rat RBL-2H3 (mast cells), and then the mRNA levels of swiprosin-1 were determined by RT-PCR. Note: Expression of swiprosin-1 in RBL-2H3 was determined by using rat swiprosin-1-specific primers. (B) Induction of swiprosin-1 in HMC-1 cells by PMA/A23187. HMC-1 cells were treated with PMA (200 nM) and A23187 (1µM). At the indicated time points, the expression of swiprosin-1 was determined by RT-PCR. (C) Induction of swiprosin-1 in RBL-2H3 cells by cross-linking of FcεRI. RBL-2H3 cells were sensitized with anti-DNP-IgE (1µg/ml) for 18 h, and then the cells were challenged with DNP-HSA (1µg/ml). At the indicated time points, the expression of swiprosin-1 was determined by RT-PCR. The information of primers (P1 and P2) was described in the Materials and Methods. Amplification of GAPDH and hTNF-α were used as an internal control and an activated mast cell marker, respectively. All data are from at least three separate experiments.

• 

  Figure 2 Ectopic expression of swiprosin-1 enhances mast cell activation by phorbol ester and calcium ionophore. (A~D) Over-expression of swiprosin-1 enhanced PMA/A23187-induced cytokine expression and histamine release. H-GFP or H-swip-1_GFP cells were treated with PMA (200 nM) for various times, and then the expression of cytokines were determined by RT-PCR (A) and quantitative RT-PCR (B). The supernatant was also used to determine IL-8 secretion (C) and histamine release (D). Amplification of GAPDH was used as an internal control. Data represent three separate experiments. The data shown in the bar graphs represent mean±SD values of triplicate experiments.

• 

  Figure 3 Inhibition of cytokine expression by various pharmacologic agents in HMC-1 cells expressing GFP or swip-1_GFP. (A, B) H-GFP or H-swip-1_GFP cells were pretreated for 30 min with or without various pharmacologic inhibitors including MG132 (1µM), cyclosporine A (CSA, 1µg/ml), PD98059 (PD, 10µM), SB203580 (SB, 10µM), wortmannin (WOT, 100 nM), PP2 (10µM), colchicines (Col, 20µM), and cytochalasin B (CTB, 1µg/ml). Then the cells were further stimulated for 4 h with or without PMA (200 nM)/A23187 (1µM). The expression of cytokines was determined by RT-PCR (top) and quantitative RT-PCR (bottom). Amplification of GAPDH was used as an internal control. Data represent three separate experiments. The data shown in the bar graphs represent mean±SD values of triplicate experiments.

• 

  Figure 4 Ectopic expression of swiprosin-1 increases phosphorylation of PI3K and Akt in HMC-1 cells. H-GFP or H-swip-1_GFP cells were treated with PMA (200 nM)/A23187 (1µM). At the indicated time points, the cells were lysed and p-PI3K and p-Akt were determined by western blot.

• 

  Figure 5 Swiprosin-1 is localized in actin-rich microvilli-like membrane protrusions in mast cells. (A) H-GFP or H-swip-1_GFP cells were adhered on glass coverslips, and then were imaged using confocal microscopy with serial z-sections. Images were represented as single series images and reconstituted images of the z-axis. (B) 293T cells were transfected with swiprosin-1/pcDNA3, then the cells were stained antiswiprosin-1 antibody, followed by incubation with the FITC-conjugated secondary antibody. The cells were then imaged by confocal microscopy. (C, D) HMC-1 cells expressing GFP or swip-1_GFP (C) or COS-7 cells expressing swip-1_GFP (D) were fixed and stained with phalloidin-TRITC. The cells were then imaged by confocal microscopy. Co-localization analysis was carried out as described in Materials and Methods. Scale bars, 10µm.

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